CLK3

CLK3 is a nuclear dual-specificity (serine/threonine and tyrosine) kinase that phosphorylates SR splicing factors to drive their redistribution from nuclear speckles and thereby regulate alternative pre-mRNA splicing (PMID:9637771). This splicing-regulatory activity operates in physiological programs: in hematopoietic stem cells CLK3 directs alternative splicing of HMGA2 to generate an isoform that escapes let-7 suppression (PMID:29625070), and in myeloid progenitors CLK3 acts as a downstream effector of DDX41 in a splicing regulatory hierarchy, with DDX41 elevating CLK3 during differentiation (PMID:41193510). Its catalytic output is constrained physiologically by polyphosphate, which inhibits CLK3 to preserve nuclear speckle stability. Beyond splicing, CLK3 directly phosphorylates USP13 at tyrosine 708, promoting USP13–c-Myc binding that blocks Fbxl14-mediated c-Myc ubiquitination, stabilizing c-Myc and reprogramming purine metabolism; a gain-of-function CLK3-Q607R mutation enhances this axis (PMID:32453420). CLK3 also functions in vertebrate neural induction during early embryogenesis (PMID:34146908). Distinct from its nuclear roles, CLK3 in mature spermatozoa localizes cytoplasmically to the acrosome and tail and is expelled and proteolytically inactivated during the acrosome reaction (PMID:10585269). CLK3 abundance is further controlled post-transcriptionally by miR-144 targeting of its 3'UTR (PMID:31807004). A CLK3-selective inhibitor (VS-77) has been developed exploiting lysine 241, unique to CLK3 among the four CLK isoforms (PMID:41158282).

1. 1996 Medium

   Established the basic catalytic behavior of CLK3 by showing the kinase autophosphorylates but is selective toward substrates, foreshadowing a dedicated rather than promiscuous activity.

   Evidence GST-fusion in vitro kinase assay on rat CLK3 with histone and casein as candidate substrates

   PMID:8679717

   Open questions at the time

   • No physiological substrate identified in this assay
   • Autophosphorylation site and regulatory consequence not defined
2. 1998 High

   Defined CLK3 as a nuclear dual-specificity kinase that controls SR protein distribution and alternative splicing, assigning it a concrete role in pre-mRNA processing.

   Evidence Overexpression of wild-type and catalytically inactive truncated isoforms with nuclear localization imaging and in vivo splicing reporter assays

   PMID:9637771

   Open questions at the time

   • Specific SR protein substrates and phospho-sites not enumerated
   • Endogenous splicing targets not identified
3. 1999 Medium

   Revealed a non-nuclear, cell-type-specific deployment of CLK3 in sperm, where it is positioned at the acrosome/tail and cleared during the acrosome reaction, indicating regulated spatial inactivation.

   Evidence Immunofluorescence in testis/epididymis sections, fractionation, Western blot, and acrosome reaction assays in mouse spermatozoa

   PMID:10585269

   Open questions at the time

   • Substrates in sperm unknown
   • Functional consequence of expulsion for fertilization not established
   • Protease responsible not identified
4. 2018 Medium

   Connected CLK3 splicing activity to stem cell biology by showing it generates an HMGA2 isoform resistant to let-7 repression, demonstrating a defined physiological splicing target.

   Evidence RNA-seq splicing analysis with CLK3 loss- and gain-of-function in human HSCs and miRNA target validation

   PMID:29625070

   Open questions at the time

   • Direct phosphorylation event driving the splice switch not mapped
   • Single lab
5. 2019 Medium

   Identified post-transcriptional control of CLK3 by miR-144 and linked CLK3 to Wnt/β-catenin signaling in hepatocellular carcinoma.

   Evidence Luciferase 3'UTR reporter assay, Western blot, RT-qPCR, and CLK3 overexpression rescue in HCC models

   PMID:31807004

   Open questions at the time

   • Mechanism linking CLK3 kinase activity to Wnt signaling not biochemically defined
   • Direct CLK3 substrates in this context unknown
6. 2020 High

   Uncovered a kinase-substrate axis beyond splicing: CLK3 phosphorylates USP13 at Y708 to stabilize c-Myc and reprogram purine metabolism, and identified a disease-associated gain-of-function mutation.

   Evidence In vitro kinase assay, mass spectrometry, co-IP, phospho-site mutagenesis, ubiquitination and reporter assays, and CLK3-Q607R mutant analysis in cholangiocarcinoma

   PMID:32453420

   Open questions at the time

   • Whether the USP13/c-Myc axis operates in normal tissues not addressed
   • Relationship to CLK3 splicing function unclear
7. 2021 Medium

   Extended CLK3 function to development, showing the ortholog is required for neural tissue formation in early vertebrate embryos.

   Evidence Morpholino knockdown, mRNA overexpression, and ectodermal explant assays with neural marker in situ hybridization in Xenopus

   PMID:34146908

   Open questions at the time

   • Molecular substrates driving neural induction not identified
   • Link to splicing vs. other activity unresolved
8. 2022 Medium

   Placed CLK3 in a cancer signaling hierarchy by showing MFAP2 stabilizes CLK3 against autophagic degradation and that CLK3 is required for MFAP2-driven invasion.

   Evidence Mass spectrometry, siRNA knockdown with CLK3 overexpression rescue, transwell invasion, and in vivo peritoneal metastasis model in colorectal cancer

   PMID:36583532

   Open questions at the time

   • Mechanism of CLK3 turnover by autophagy not detailed
   • Direct CLK3 effectors in invasion unknown
9. 2024 Low

   Associated CLK3 with colorectal cancer proliferation through proposed JAK2/STAT3 and c-MYC routes, though mechanistic detail rests on expression and stability readouts.

   Evidence CLK3 knockdown/overexpression, Western blot, in vitro proliferation, and xenograft assays

   PMID:38885806 PMID:39289754

   Open questions at the time

   • JAK2 stabilization mechanism shown by Western blot alone without direct biochemistry
   • CLK3→c-MYC link inferred from expression changes, not direct phosphorylation
10. 2025 Medium

    Identified physiological regulation of CLK3 catalysis by polyphosphate and embedded CLK3 in a DDX41-governed splicing hierarchy, refining how CLK3 splicing output is controlled.

    Evidence Proximity biotinylation, polyP kinase inhibition and SR phosphorylation assays with RNA-seq (preprint, bioRxiv); Ddx41+/- model with global RNA-seq splicing and CLK3 loss-of-function in myeloid progenitors

    PMID:41193510

    Open questions at the time

    • polyP findings are preprint and single lab
    • How DDX41 elevates CLK3 transcript mechanistically not defined
    • Which splicing events require CLK3 vs. not is incompletely mapped
11. 2025 Medium

    Enabled isoform-selective targeting by identifying CLK3-unique lysine 241 and developing a selective inhibitor, providing a chemical tool to dissect CLK3-specific functions.

    Evidence Structure-guided active-site analysis, medicinal chemistry, and in vitro IC50 determination for VS-77

    PMID:41158282

    Open questions at the time

    • Role of K241 not validated by mutagenesis
    • Cellular selectivity and efficacy not established

• It remains unresolved how CLK3's splicing-regulatory activity, its USP13/c-Myc phosphorylation axis, and its developmental and sperm roles are integrated, and which endogenous substrates govern each context.
• No unified substrate map across contexts
• Determinants of nuclear vs. cytoplasmic deployment unknown
• Physiological vs. oncogenic functions not clearly separated